anti dnaj homolog  (Proteintech)

Journal: American Journal of Cancer Research

Article Title: CCT5 as a candidate biomarker in bladder cancer: functional validation and mechanistic clues

doi: 10.62347/EBFE5165

Figure Lengend Snippet: Upregulation of CCT5 was observed in bladder cancer. A, B. Pan-cancer analysis of CCT5 expression across 33 tumor types from the TCGA database. C, D. Evaluation of CCT5 expression in paired and unpaired samples from TCGA-BLCA cohorts. E, F. Validation of elevated CCT5 expression in BLCA using GSE7476 and GSE13507 . G. Immunohistochemical staining of clinical specimens reveals stronger CCT5 expression in bladder tumor tissues compared to normal urothelium. H. RT-qPCR results demonstrating upregulation of CCT5 in bladder carcinoma tissues relative to matched adjacent non-cancerous tissues. I, J. RT-qPCR and Western blot analyses confirm higher CCT5 levels in UMUC-3, 5637, J82, T24, and 253J than in the SV-HUC-1.

Article Snippet: Sections were incubated overnight at 4°C with primary antibodies against CCT5 (1:200, Proteintech, 11603-1-AP) and Ki67 (1:1000, Proteintech, 27309-1-AP).

Techniques: Expressing, Biomarker Discovery, Immunohistochemical staining, Staining, Quantitative RT-PCR, Western Blot

Journal: American Journal of Cancer Research

Article Title: CCT5 as a candidate biomarker in bladder cancer: functional validation and mechanistic clues

doi: 10.62347/EBFE5165

Figure Lengend Snippet: High CCT5 expression correlates with poor survival and aggressive clinical features. A-C. Kaplan-Meier curves revealed that high CCT5 expression was correlated with adverse survival outcomes. D, E. Differential expression of CCT5 across distinct stages of BLCA. F, G. Expression variations of CCT5 between papillary and non-papillary subtypes, as well as between low-grade and high-grade BLCA. H. ROC analysis underscored the strong diagnostic capacity of CCT5 (AUC = 0.835).

Article Snippet: Sections were incubated overnight at 4°C with primary antibodies against CCT5 (1:200, Proteintech, 11603-1-AP) and Ki67 (1:1000, Proteintech, 27309-1-AP).

Techniques: Expressing, Quantitative Proteomics, Diagnostic Assay

Journal: American Journal of Cancer Research

Article Title: CCT5 as a candidate biomarker in bladder cancer: functional validation and mechanistic clues

doi: 10.62347/EBFE5165

Figure Lengend Snippet: Functional enrichment analysis of CCT5-related genes in BLCA. A. Volcano plot of DEGs between the CCT5-high and CCT5-low groups in the TCGA-BLCA cohort. B. Heatmap of the top 20 positively correlated genes with CCT5 in the TCGA-BLCA cohort. C-E. GO enrichment analysis of CCT5-co-expressed genes in BP, CC, and MF. F. KEGG analysis showed enrichment in the cell cycle pathway. G, H. GSEA linked high CCT5 expression to dysregulation of bladder cancer and Hippo signaling pathways.

Article Snippet: Sections were incubated overnight at 4°C with primary antibodies against CCT5 (1:200, Proteintech, 11603-1-AP) and Ki67 (1:1000, Proteintech, 27309-1-AP).

Techniques: Functional Assay, Expressing, Protein-Protein interactions

Journal: American Journal of Cancer Research

Article Title: CCT5 as a candidate biomarker in bladder cancer: functional validation and mechanistic clues

doi: 10.62347/EBFE5165

Figure Lengend Snippet: Cell-type distribution of CCT5 at the single-cell level and external validation. A, B. UMAP plot of major cell populations. C. Dot plot of canonical marker genes for cell annotation. D. CCT5 enrichment in epithelial cells. E. Stacked bar plot showing cell-type composition across samples. F. CCT5 expression across cell clusters.

Article Snippet: Sections were incubated overnight at 4°C with primary antibodies against CCT5 (1:200, Proteintech, 11603-1-AP) and Ki67 (1:1000, Proteintech, 27309-1-AP).

Techniques: Single Cell, Biomarker Discovery, Marker, Expressing

Journal: American Journal of Cancer Research

Article Title: CCT5 as a candidate biomarker in bladder cancer: functional validation and mechanistic clues

doi: 10.62347/EBFE5165

Figure Lengend Snippet: Inferred CNV features and functional associations of CCT5 based on single-cell data. A, B. InferCNV and violin plot showing higher CNV in CCT5-high cells. C. Pathway enrichment of DEGs from CCT5-high epithelial cells. D. Spatial transcriptomics showing CCT5 distribution in tumor areas. E. Cell communication between CCT5-high epithelial and other cell types.

Article Snippet: Sections were incubated overnight at 4°C with primary antibodies against CCT5 (1:200, Proteintech, 11603-1-AP) and Ki67 (1:1000, Proteintech, 27309-1-AP).

Techniques: Functional Assay, Single Cell, Spatial Transcriptomics

Journal: American Journal of Cancer Research

Article Title: CCT5 as a candidate biomarker in bladder cancer: functional validation and mechanistic clues

doi: 10.62347/EBFE5165

Figure Lengend Snippet: Effects of CCT5 knockdown on malignant phenotypes. A-D. Efficient knockdown of CCT5 in UMUC-3 and T24 cells was validated by Western blot and RT-qPCR. E, F. Proliferation capacity was compromised in CCT5-depleted cells (shCCT5 groups) as determined by CCK-8 assay. G. Clonogenic survival was markedly attenuated upon CCT5 knockdown, evidenced by a reduction in colony numbers. H, I. Transwell assays revealed impaired migratory and invasive capabilities in CCT5-silenced cells. J, K. Wound healing progression was delayed following CCT5 ablation, indicating suppressed migration.

Article Snippet: Sections were incubated overnight at 4°C with primary antibodies against CCT5 (1:200, Proteintech, 11603-1-AP) and Ki67 (1:1000, Proteintech, 27309-1-AP).

Techniques: Knockdown, Western Blot, Quantitative RT-PCR, CCK-8 Assay, Migration

Journal: American Journal of Cancer Research

Article Title: CCT5 as a candidate biomarker in bladder cancer: functional validation and mechanistic clues

doi: 10.62347/EBFE5165

Figure Lengend Snippet: Knockdown of CCT5 results in enhanced apoptotic activity and cell cycle inhibition. A, B. Flow cytometric analysis revealed a significant increase in apoptosis upon CCT5 knockdown in UMUC-3 and T24 cells. C, D. CCT5 depletion induced cell cycle arrest at the G1 phase, accompanied by a reduction in S and G2/M phase populations. E, F. Western blotting confirmed concomitant downregulation of key G1/S checkpoint regulators (Cyclin D1, CDK4, CDK6) in CCT5-deficient cells.

Article Snippet: Sections were incubated overnight at 4°C with primary antibodies against CCT5 (1:200, Proteintech, 11603-1-AP) and Ki67 (1:1000, Proteintech, 27309-1-AP).

Techniques: Knockdown, Activity Assay, Inhibition, Western Blot

Journal: American Journal of Cancer Research

Article Title: CCT5 as a candidate biomarker in bladder cancer: functional validation and mechanistic clues

doi: 10.62347/EBFE5165

Figure Lengend Snippet: CCT5 overexpression drives malignant progression. A, B. Overexpression of CCT5 in J82 cells was confirmed by RT-qPCR and Western blot. C, D. Enhanced proliferative and clonogenic capacity was observed upon CCT5 overexpression, as assessed by CCK-8 and colony formation assays. E, F. Migration, invasion, and wound healing assays demonstrated promoted migratory ability in CCT5-overexpressing cells. G. Flow cytometry indicated that CCT5 overexpression attenuated apoptosis. H, I. Cell cycle analysis and Western blot revealed that CCT5 overexpression accelerated G1/S transition and upregulated expression of Cyclin D1, CDK4, and CDK6.

Article Snippet: Sections were incubated overnight at 4°C with primary antibodies against CCT5 (1:200, Proteintech, 11603-1-AP) and Ki67 (1:1000, Proteintech, 27309-1-AP).

Techniques: Over Expression, Quantitative RT-PCR, Western Blot, CCK-8 Assay, Migration, Flow Cytometry, Cell Cycle Assay, Expressing

Journal: American Journal of Cancer Research

Article Title: CCT5 as a candidate biomarker in bladder cancer: functional validation and mechanistic clues

doi: 10.62347/EBFE5165

Figure Lengend Snippet: CCT5 knockdown suppresses tumor growth in vivo and activates Hippo signaling. A-C. Subcutaneous xenograft models demonstrated impaired tumor growth upon CCT5 knockdown. D. Tumors with CCT5 knockdown exhibited lower proliferative index, as indicated by reduced Ki-67 staining. E, F. Transcriptomic profiling revealed downregulation of Hippo pathway downstream effectors (e.g., BMP4, CCN1, AMOT, CCN2). G, H. GO and KEGG analyses of differentially expressed genes highlighted enrichments in processes including wound healing, cell development, and Hippo signaling. I, J. Western blot analysis confirmed activation of the Hippo pathway, evidenced by enhanced phosphorylation of MST1, LATS1, and YAP without alterations in total protein levels.

Article Snippet: Sections were incubated overnight at 4°C with primary antibodies against CCT5 (1:200, Proteintech, 11603-1-AP) and Ki67 (1:1000, Proteintech, 27309-1-AP).

Techniques: Knockdown, In Vivo, Staining, Western Blot, Activation Assay, Phospho-proteomics

Journal: American Journal of Cancer Research

Article Title: CCT5 as a candidate biomarker in bladder cancer: functional validation and mechanistic clues

doi: 10.62347/EBFE5165

Figure Lengend Snippet: Upregulation of CCT5 was observed in bladder cancer. A, B. Pan-cancer analysis of CCT5 expression across 33 tumor types from the TCGA database. C, D. Evaluation of CCT5 expression in paired and unpaired samples from TCGA-BLCA cohorts. E, F. Validation of elevated CCT5 expression in BLCA using GSE7476 and GSE13507 . G. Immunohistochemical staining of clinical specimens reveals stronger CCT5 expression in bladder tumor tissues compared to normal urothelium. H. RT-qPCR results demonstrating upregulation of CCT5 in bladder carcinoma tissues relative to matched adjacent non-cancerous tissues. I, J. RT-qPCR and Western blot analyses confirm higher CCT5 levels in UMUC-3, 5637, J82, T24, and 253J than in the SV-HUC-1.

Article Snippet: The following antibodies were used: GAPDH (1:1000; Servicebio, #GB15004-100), CCT5 (1:1000; Proteintech, #11603-1-AP), CDK4 (1:1000; Proteintech, #11026-1-AP), CDK6 (1:1000; Proteintech, #14052-1-AP), Cyclin D1 (1:1000; Proteintech, #26939-1-AP), phospho-MST1/2 (1:1000; Cell Signaling Technology, #49332), MST1 (1:500; Santa Cruz, #sc-515051), LATS1 (1:500; Santa Cruz, #sc-398560), phospho-LATS1 (1:1000; Cell Signaling Technology, #8654), YAP1 (1:500; Santa Cruz, #sc-101199), and phospho-YAP1 (1:1000; Abways, #CY5743).

Techniques: Expressing, Biomarker Discovery, Immunohistochemical staining, Staining, Quantitative RT-PCR, Western Blot

Journal: American Journal of Cancer Research

Article Title: CCT5 as a candidate biomarker in bladder cancer: functional validation and mechanistic clues

doi: 10.62347/EBFE5165

Figure Lengend Snippet: High CCT5 expression correlates with poor survival and aggressive clinical features. A-C. Kaplan-Meier curves revealed that high CCT5 expression was correlated with adverse survival outcomes. D, E. Differential expression of CCT5 across distinct stages of BLCA. F, G. Expression variations of CCT5 between papillary and non-papillary subtypes, as well as between low-grade and high-grade BLCA. H. ROC analysis underscored the strong diagnostic capacity of CCT5 (AUC = 0.835).

Article Snippet: The following antibodies were used: GAPDH (1:1000; Servicebio, #GB15004-100), CCT5 (1:1000; Proteintech, #11603-1-AP), CDK4 (1:1000; Proteintech, #11026-1-AP), CDK6 (1:1000; Proteintech, #14052-1-AP), Cyclin D1 (1:1000; Proteintech, #26939-1-AP), phospho-MST1/2 (1:1000; Cell Signaling Technology, #49332), MST1 (1:500; Santa Cruz, #sc-515051), LATS1 (1:500; Santa Cruz, #sc-398560), phospho-LATS1 (1:1000; Cell Signaling Technology, #8654), YAP1 (1:500; Santa Cruz, #sc-101199), and phospho-YAP1 (1:1000; Abways, #CY5743).

Techniques: Expressing, Quantitative Proteomics, Diagnostic Assay

Journal: American Journal of Cancer Research

Article Title: CCT5 as a candidate biomarker in bladder cancer: functional validation and mechanistic clues

doi: 10.62347/EBFE5165

Figure Lengend Snippet: Functional enrichment analysis of CCT5-related genes in BLCA. A. Volcano plot of DEGs between the CCT5-high and CCT5-low groups in the TCGA-BLCA cohort. B. Heatmap of the top 20 positively correlated genes with CCT5 in the TCGA-BLCA cohort. C-E. GO enrichment analysis of CCT5-co-expressed genes in BP, CC, and MF. F. KEGG analysis showed enrichment in the cell cycle pathway. G, H. GSEA linked high CCT5 expression to dysregulation of bladder cancer and Hippo signaling pathways.

Article Snippet: The following antibodies were used: GAPDH (1:1000; Servicebio, #GB15004-100), CCT5 (1:1000; Proteintech, #11603-1-AP), CDK4 (1:1000; Proteintech, #11026-1-AP), CDK6 (1:1000; Proteintech, #14052-1-AP), Cyclin D1 (1:1000; Proteintech, #26939-1-AP), phospho-MST1/2 (1:1000; Cell Signaling Technology, #49332), MST1 (1:500; Santa Cruz, #sc-515051), LATS1 (1:500; Santa Cruz, #sc-398560), phospho-LATS1 (1:1000; Cell Signaling Technology, #8654), YAP1 (1:500; Santa Cruz, #sc-101199), and phospho-YAP1 (1:1000; Abways, #CY5743).

Techniques: Functional Assay, Expressing, Protein-Protein interactions

Journal: American Journal of Cancer Research

Article Title: CCT5 as a candidate biomarker in bladder cancer: functional validation and mechanistic clues

doi: 10.62347/EBFE5165

Figure Lengend Snippet: Cell-type distribution of CCT5 at the single-cell level and external validation. A, B. UMAP plot of major cell populations. C. Dot plot of canonical marker genes for cell annotation. D. CCT5 enrichment in epithelial cells. E. Stacked bar plot showing cell-type composition across samples. F. CCT5 expression across cell clusters.

Article Snippet: The following antibodies were used: GAPDH (1:1000; Servicebio, #GB15004-100), CCT5 (1:1000; Proteintech, #11603-1-AP), CDK4 (1:1000; Proteintech, #11026-1-AP), CDK6 (1:1000; Proteintech, #14052-1-AP), Cyclin D1 (1:1000; Proteintech, #26939-1-AP), phospho-MST1/2 (1:1000; Cell Signaling Technology, #49332), MST1 (1:500; Santa Cruz, #sc-515051), LATS1 (1:500; Santa Cruz, #sc-398560), phospho-LATS1 (1:1000; Cell Signaling Technology, #8654), YAP1 (1:500; Santa Cruz, #sc-101199), and phospho-YAP1 (1:1000; Abways, #CY5743).

Techniques: Single Cell, Biomarker Discovery, Marker, Expressing

Journal: American Journal of Cancer Research

Article Title: CCT5 as a candidate biomarker in bladder cancer: functional validation and mechanistic clues

doi: 10.62347/EBFE5165

Figure Lengend Snippet: Inferred CNV features and functional associations of CCT5 based on single-cell data. A, B. InferCNV and violin plot showing higher CNV in CCT5-high cells. C. Pathway enrichment of DEGs from CCT5-high epithelial cells. D. Spatial transcriptomics showing CCT5 distribution in tumor areas. E. Cell communication between CCT5-high epithelial and other cell types.

Article Snippet: The following antibodies were used: GAPDH (1:1000; Servicebio, #GB15004-100), CCT5 (1:1000; Proteintech, #11603-1-AP), CDK4 (1:1000; Proteintech, #11026-1-AP), CDK6 (1:1000; Proteintech, #14052-1-AP), Cyclin D1 (1:1000; Proteintech, #26939-1-AP), phospho-MST1/2 (1:1000; Cell Signaling Technology, #49332), MST1 (1:500; Santa Cruz, #sc-515051), LATS1 (1:500; Santa Cruz, #sc-398560), phospho-LATS1 (1:1000; Cell Signaling Technology, #8654), YAP1 (1:500; Santa Cruz, #sc-101199), and phospho-YAP1 (1:1000; Abways, #CY5743).

Techniques: Functional Assay, Single Cell, Spatial Transcriptomics

Journal: American Journal of Cancer Research

Article Title: CCT5 as a candidate biomarker in bladder cancer: functional validation and mechanistic clues

doi: 10.62347/EBFE5165

Figure Lengend Snippet: Effects of CCT5 knockdown on malignant phenotypes. A-D. Efficient knockdown of CCT5 in UMUC-3 and T24 cells was validated by Western blot and RT-qPCR. E, F. Proliferation capacity was compromised in CCT5-depleted cells (shCCT5 groups) as determined by CCK-8 assay. G. Clonogenic survival was markedly attenuated upon CCT5 knockdown, evidenced by a reduction in colony numbers. H, I. Transwell assays revealed impaired migratory and invasive capabilities in CCT5-silenced cells. J, K. Wound healing progression was delayed following CCT5 ablation, indicating suppressed migration.

Article Snippet: The following antibodies were used: GAPDH (1:1000; Servicebio, #GB15004-100), CCT5 (1:1000; Proteintech, #11603-1-AP), CDK4 (1:1000; Proteintech, #11026-1-AP), CDK6 (1:1000; Proteintech, #14052-1-AP), Cyclin D1 (1:1000; Proteintech, #26939-1-AP), phospho-MST1/2 (1:1000; Cell Signaling Technology, #49332), MST1 (1:500; Santa Cruz, #sc-515051), LATS1 (1:500; Santa Cruz, #sc-398560), phospho-LATS1 (1:1000; Cell Signaling Technology, #8654), YAP1 (1:500; Santa Cruz, #sc-101199), and phospho-YAP1 (1:1000; Abways, #CY5743).

Techniques: Knockdown, Western Blot, Quantitative RT-PCR, CCK-8 Assay, Migration

Journal: American Journal of Cancer Research

Article Title: CCT5 as a candidate biomarker in bladder cancer: functional validation and mechanistic clues

doi: 10.62347/EBFE5165

Figure Lengend Snippet: Knockdown of CCT5 results in enhanced apoptotic activity and cell cycle inhibition. A, B. Flow cytometric analysis revealed a significant increase in apoptosis upon CCT5 knockdown in UMUC-3 and T24 cells. C, D. CCT5 depletion induced cell cycle arrest at the G1 phase, accompanied by a reduction in S and G2/M phase populations. E, F. Western blotting confirmed concomitant downregulation of key G1/S checkpoint regulators (Cyclin D1, CDK4, CDK6) in CCT5-deficient cells.

Article Snippet: The following antibodies were used: GAPDH (1:1000; Servicebio, #GB15004-100), CCT5 (1:1000; Proteintech, #11603-1-AP), CDK4 (1:1000; Proteintech, #11026-1-AP), CDK6 (1:1000; Proteintech, #14052-1-AP), Cyclin D1 (1:1000; Proteintech, #26939-1-AP), phospho-MST1/2 (1:1000; Cell Signaling Technology, #49332), MST1 (1:500; Santa Cruz, #sc-515051), LATS1 (1:500; Santa Cruz, #sc-398560), phospho-LATS1 (1:1000; Cell Signaling Technology, #8654), YAP1 (1:500; Santa Cruz, #sc-101199), and phospho-YAP1 (1:1000; Abways, #CY5743).

Techniques: Knockdown, Activity Assay, Inhibition, Western Blot

Journal: American Journal of Cancer Research

Article Title: CCT5 as a candidate biomarker in bladder cancer: functional validation and mechanistic clues

doi: 10.62347/EBFE5165

Figure Lengend Snippet: CCT5 overexpression drives malignant progression. A, B. Overexpression of CCT5 in J82 cells was confirmed by RT-qPCR and Western blot. C, D. Enhanced proliferative and clonogenic capacity was observed upon CCT5 overexpression, as assessed by CCK-8 and colony formation assays. E, F. Migration, invasion, and wound healing assays demonstrated promoted migratory ability in CCT5-overexpressing cells. G. Flow cytometry indicated that CCT5 overexpression attenuated apoptosis. H, I. Cell cycle analysis and Western blot revealed that CCT5 overexpression accelerated G1/S transition and upregulated expression of Cyclin D1, CDK4, and CDK6.

Article Snippet: The following antibodies were used: GAPDH (1:1000; Servicebio, #GB15004-100), CCT5 (1:1000; Proteintech, #11603-1-AP), CDK4 (1:1000; Proteintech, #11026-1-AP), CDK6 (1:1000; Proteintech, #14052-1-AP), Cyclin D1 (1:1000; Proteintech, #26939-1-AP), phospho-MST1/2 (1:1000; Cell Signaling Technology, #49332), MST1 (1:500; Santa Cruz, #sc-515051), LATS1 (1:500; Santa Cruz, #sc-398560), phospho-LATS1 (1:1000; Cell Signaling Technology, #8654), YAP1 (1:500; Santa Cruz, #sc-101199), and phospho-YAP1 (1:1000; Abways, #CY5743).

Techniques: Over Expression, Quantitative RT-PCR, Western Blot, CCK-8 Assay, Migration, Flow Cytometry, Cell Cycle Assay, Expressing

Journal: American Journal of Cancer Research

Article Title: CCT5 as a candidate biomarker in bladder cancer: functional validation and mechanistic clues

doi: 10.62347/EBFE5165

Figure Lengend Snippet: CCT5 knockdown suppresses tumor growth in vivo and activates Hippo signaling. A-C. Subcutaneous xenograft models demonstrated impaired tumor growth upon CCT5 knockdown. D. Tumors with CCT5 knockdown exhibited lower proliferative index, as indicated by reduced Ki-67 staining. E, F. Transcriptomic profiling revealed downregulation of Hippo pathway downstream effectors (e.g., BMP4, CCN1, AMOT, CCN2). G, H. GO and KEGG analyses of differentially expressed genes highlighted enrichments in processes including wound healing, cell development, and Hippo signaling. I, J. Western blot analysis confirmed activation of the Hippo pathway, evidenced by enhanced phosphorylation of MST1, LATS1, and YAP without alterations in total protein levels.

Article Snippet: The following antibodies were used: GAPDH (1:1000; Servicebio, #GB15004-100), CCT5 (1:1000; Proteintech, #11603-1-AP), CDK4 (1:1000; Proteintech, #11026-1-AP), CDK6 (1:1000; Proteintech, #14052-1-AP), Cyclin D1 (1:1000; Proteintech, #26939-1-AP), phospho-MST1/2 (1:1000; Cell Signaling Technology, #49332), MST1 (1:500; Santa Cruz, #sc-515051), LATS1 (1:500; Santa Cruz, #sc-398560), phospho-LATS1 (1:1000; Cell Signaling Technology, #8654), YAP1 (1:500; Santa Cruz, #sc-101199), and phospho-YAP1 (1:1000; Abways, #CY5743).

Techniques: Knockdown, In Vivo, Staining, Western Blot, Activation Assay, Phospho-proteomics

Journal: The Journal of Clinical Investigation

Article Title: ANKRD55 is a key regulator of T cell inflammation in multiple sclerosis

doi: 10.1172/JCI195214

Figure Lengend Snippet: ( A ) Overexpression of ANKRD55 and TCP1 in HEK293T cells followed by co-IP analysis to determine their interaction. ( B ) Protein extraction from Jurkat cells with subsequent co-IP assay to determine the interaction between ANKRD55 and TCP1. ( C ) PLA experiment in Jurkat cells to assess the interaction between ANKRD55 and TCP1. Cells were fixed and incubated with primary antibodies against ANKRD55 and TCP1, followed by PLA probe ligation and amplification. Red fluorescent puncta indicate close proximity (<40 nm) between ANKRD55 and TCP1, suggesting a direct or complex-mediated interaction. Nuclei were counterstained with DAPI (blue). Representative images are shown. Scale bars: 2 μm (top), 5 μm (bottom). ( D ) Immunofluorescent staining of Jurkat cells to analyze the colocalization of ANKRD55 and TCP1. Scale bars: 2 μm. ( E – K ) Co-IP assays were performed using HEK293T cells to investigate the interactions between ANKRD55 and individual CCT subunits, including CCT2, CCT3, CCT4, CCT5, CCT6, CCT7, and CCT8. ( L ) Immunofluorescent staining of Jurkat cells to assess colocalization between ANKRD55 and specific CCT subunits (CCT2, CCT3, CCT4, and CCT7). Scale bars: 2 μm (first 4 panels), 1 μm (fifth panel). ( M ) Immunofluorescent staining of Jurkat cells to visualize the subcellular localization of ANKRD55, TCP1, and pericentrin. Scale bars: 5 μm. ( N – P ) Immunoblotting analysis of TCP1 ( N ), CCT3 ( O ), and CCT6 ( P ) expression levels in Jurkat cells following ANKRD55 knockdown.

Article Snippet: TCP1 antibody (catalog 10320 and 68183), CCT2 antibody (catalog 68214), CCT3 antibody (catalog 60264), CCT4 antibody (catalog 67455), CCT5 antibody (catalog 67400), CCT6 antibody (catalog 19793), CCT7 antibody (catalog 68214), DYKDDDDK tag polyclonal antibody (catalog 20543), HA tag polyclonal antibody (catalog 51064), phospho-ERK1/2 (Thr202/Tyr204) polyclonal antibody (catalog 28733), CD247 polyclonal antibody (catalog 12837), and phospho-LCK-Y394 rabbit antibody (catalog AP0182) were purchased from Proteintech.

Techniques: Over Expression, Co-Immunoprecipitation Assay, Protein Extraction, Incubation, Ligation, Amplification, Staining, Western Blot, Expressing, Knockdown

Journal: The Journal of Clinical Investigation

Article Title: ANKRD55 is a key regulator of T cell inflammation in multiple sclerosis

doi: 10.1172/JCI195214

Figure Lengend Snippet: ( A and B ) α-Tubulin immunoblotting in lysates and pellet determined by microtubule sedimentation assay in Jurkat cells with TCP1 ( A ) or ANKRD55 ( B ) knocked down. ( C ) TCP1 degradation rate analyzed via immunoblot after cycloheximide (CHX; 70 μM) treatment for 0–48 hours in control and Jurkat cells overexpressing ANKRD55. ( D – F ) Co-IP detection of interactions between CCT5 and TCP1 ( D ), CCT3 ( E ), or CCT6 ( F ) at varying concentrations of ANKRD55 in HEK293T. ( G ) Immunofluorescence analysis of immune synapse formation between Jurkat and Raji cells. Jurkat cells were prelabeled with CMAC. Raji cells were stimulated with SEE for 30 minutes. The 2 cell types were then cocultured for 30 minutes. Cells were stained with antibodies against ANKRD55, TCP1, pericentrin, and α-tubulin to visualize protein localization at the immune synapse. Scale bars: 2 μm. BF, bright-field; CMAC, CellTracker blue fluorescent probe. ( H ) Flow cytometry–based immune synapse (IS) pattern analysis. ( I and J ) Raji cells (APCs) stained with CFSE and stimulated with SEE for 30 minutes at 37°C and Jurkat cells (T cells) stained with CMTPX. T cell conjugation with APCs after 20 minutes of contact was analyzed by flow cytometry. Conjugate percentages were determined for Jurkat cells with ANKRD55 or TCP1 knocked down ( I ) and pretreatment with HSF1A (50 μM) for 2 hours ( J ). ( K ) Mean clinical score of EAE in mice injected intraperitoneally with PBS or HSF1A (20 mg/mL) ( n = 7 or 8 mice per group), induced by active immunization with MOG 35–55 . ( L ) Immunoblot analysis of TCR signaling in Jurkat cells. Cells included vector control, a stable ANKRD55-overexpressing cell line, and ANKRD55-overexpressing cells pretreated with HSF1A (50 μM, 2 hours). All groups were stimulated on plates coated with anti-CD3 and anti-CD28. Lysates were collected at the indicated time points (1, 2, 15, and 30 minutes) and probed for TCR signaling–associated proteins. ( M ) H&E and Luxol fast blue (LFB) staining of spinal cord sections at the peak of EAE disease. Arrows indicate areas of demyelination. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, by 2-way ANOVA with Tukey’s multiple-comparison test. Data are shown as mean ± SEM.

Article Snippet: TCP1 antibody (catalog 10320 and 68183), CCT2 antibody (catalog 68214), CCT3 antibody (catalog 60264), CCT4 antibody (catalog 67455), CCT5 antibody (catalog 67400), CCT6 antibody (catalog 19793), CCT7 antibody (catalog 68214), DYKDDDDK tag polyclonal antibody (catalog 20543), HA tag polyclonal antibody (catalog 51064), phospho-ERK1/2 (Thr202/Tyr204) polyclonal antibody (catalog 28733), CD247 polyclonal antibody (catalog 12837), and phospho-LCK-Y394 rabbit antibody (catalog AP0182) were purchased from Proteintech.

Techniques: Western Blot, Microtubule Sedimentation Assay, Control, Co-Immunoprecipitation Assay, Immunofluorescence, Staining, Flow Cytometry, Conjugation Assay, Injection, Plasmid Preparation, Comparison